Abstract Staphylococcus aureus is involved in ~80% of all orthopaedic infections. Although the incidence of infections following elective surgery is low (<3%), reinfection rates are very high (15-40%), which has led to the orthopaedic paradigm that S. aureus infection of bone is incurable. This problem is exacerbated in patients with infected diabetic foot ulcers (DFU), in which recurrent infections can lead to lower limb amputation or septic death. The cost in morbidity, mortality, and time lost from work and family is enormous. Currently, the pathogenic mechanisms responsible for the unique problems of osteomyelitis relative to bacterial colonization of soft tissues or foreign bodies remain unknown. To address this, we completed the first extensive transmission electron microcopy (TEM) study of S. aureus infected cortical bone , which produced several remarkable findings. Most notable was S. aureus colonization of canaliculi and submicron channels of live bone, suggesting that even the most extensive debridement would be unable to remove these bacteria during revision surgery. More recently, we have identified similar S. aureus colonization of osteocytic canalicular networks in amputated bone from patients with infected DFU. These novel findings challenge the dogma that S. aureus are non-motile cocci, and have ramifications for treatment strategies to eradicate monomicrobial and polymicrobial osteomyelitis. Therefore, follow up studies are warranted to test the hypotheses that: 1) the high prevalence of S. aureus in osteomyelitis is partially due to its unique ability to colonize osteocytic canalicular networks; and 2) susceptible strains of S. aureus (based on in vitro minimal inhibitory concentration (MIC)) become resistant to ?standard-of-care? dosing regimens of antibiotics following bone infection due to inferior biodistribution and pharmacokinetics of the drugs within osteocytic canalicular networks relative to the soft tissue abscesses they were designed to treat. To test this, we propose two Specific Aims. In Aim 1 we will assess the incidence of S. aureus colonization of osteocytic canalicular networks in amputated bone. DFU patients with clinical culture confirmed monomicrobial (S. aureus only) and polymicrobial (S. aureus and one or more other bacterial species), who are scheduled to undergo a toe amputation surgery, will be recruited from the PI's clinical practice. The discarded bone tissue samples will be processed for immunoelectron microscopy (IEM) using S. aureus specific antibodies to determine the incidence and composition of the bacteria within canaliculi of the infected bone. In Aim 2 we will assess the ability of standard of care antibiotic therapy to eradicated S. aureus in canaliculi versus adjacent soft tissue in an established murine model of implant associated osteomyelitis. While our novel findings of S. aureus colonization of canaliculi in experimental and clinical osteomyelitis challenges established paradigms of bone infection and patient care, the proposed research is needed to formally confirm these initial observations towards novel interventions for DFU patients.